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System for in vitro transposition using modified TN5 transposase

A technology of transposase, donor, application in the system, 10 Tr, trand-Transfer Reaction at t, field of production of modified transposase

Inactive Publication Date: 2004-08-25
WISCONSIN ALUMNI RES FOUND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, the ends of the Tn10 element limit its use in general in vitro transposition systems

Method used

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  • System for in vitro transposition using modified TN5 transposase
  • System for in vitro transposition using modified TN5 transposase
  • System for in vitro transposition using modified TN5 transposase

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Embodiment

[0063] To obtain the modified transposase at position 54, the first three coding regions of an existing DNA clone encoding the Tn5 transposase (instead of the inhibitor protein MA56) were mutagenized according to known methods, Partial DNA fragments were cloned to generate a library of plasmid clones containing the full-length transposase gene. The clones constituting the library were transformed into Escherichia coli K-12 strain MDW320 bacteria, and the bacteria were inoculated for plate culture to grow into colonies. The transposable element provided on a plasmid isolated in bacteria contains a lacZ-deficient gene. The isolated plasmid pOXgen386 is described in Weinreich, M. et al., "A functional analysis of the Tn5 Transposase: Identification of Domains Required for DNA Binding and Dimerization," J. Mol. Biol .241:166-177 (1993), the contents of which are incorporated herein by reference. Colonies with increased transposase activity were selected by screening for blue ...

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Abstract

A system for in vitro transposition includes a donor DNA that includes a transposable element flanked by a pair of bacterial transposon Tn5 outside end repeat sequences, a target DNA into which the transposable element can transpose, and a modified Tn5 transposase having higher binding avidity to the outside end repeat sequences and being less likely to assume an inactive multimer form than wild type Tn5 transposase.

Description

[0001] Cross References to Related Applications [0002] This patent application is a partial continuation application of the patent application entitled "In Vitro Transposition System", which was filed on March 11, 1997, and has not yet been issued a patent number. Applicants have requested September 9, 1996 as the filing date of this patent application. [0003] Statement Regarding Federally Sponsored Research or Development [0004] Not applicable. Background of the invention [0005] The present invention relates generally to the field of transposable nucleic acids, and more particularly to the production of modified transposases and their use in systems for introducing genetic changes into nucleic acids. [0006] Transposable genetic elements are DNA sequences found in various prokaryotic and eukaryotic organisms that are capable of moving or switching from one location in the genome to another. In vivo, trans...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/09C12N9/22C12N15/55C12N15/85C12N15/90
CPCC12N2800/90C12N15/90C12N9/22C12N15/85
Inventor W·S·列兹尼科夫I·Y·戈雷申周红
Owner WISCONSIN ALUMNI RES FOUND